Correlated Occurrence and Bypass of Frame-Shifting Insertion-Deletions (InDels) to Give Functional Proteins

Abstract

Short insertions and deletions (InDels) comprise an important part of the natural mutational repertoire. InDels are, however, highly deleterious, primarily because two-thirds result in frame-shifts. Bypass through slippage over homonucleotide repeats by transcriptional and/or translational infidelity is known to occur sporadically. However, the overall frequency of bypass and its relation to sequence composition remain unclear. Intriguingly, the occurrence of InDels and the bypass of frame-shifts are mechanistically related - occurring through slippage over repeats by DNA or RNA polymerases, or by the ribosome, respectively. Here, we show that the frequency of frame-shifting InDels, and the frequency by which they are bypassed to give full-length, functional proteins, are indeed highly correlated. Using a laboratory genetic drift, we have exhaustively mapped all InDels that occurred within a single gene. We thus compared the naive InDel repertoire that results from DNA polymerase slippage to the frame-shifting InDels tolerated following selection to maintain protein function. We found that InDels repeatedly occurred, and were bypassed, within homonucleotide repeats of 3–8 bases. The longer the repeat, the higher was the frequency of InDels formation, and the more frequent was their bypass. Besides an expected 8A repeat, other types of repeats, including short ones, and G and C repeats, were bypassed. Although obtained in vitro, our results indicate a direct link between the genetic occurrence of InDels and their phenotypic rescue, thus suggesting a potential role for frame-shifting InDels as bridging evolutionary intermediates.

Author Summary

Homonucleotide repeats are exceptionally prone to insertions and/or deletions of bases (InDels). However, unless they occur in a multiplicity of 3 bases, InDels disrupt the reading frame and are thus expected to be purged from coding regions. Homonucleotide repeats, however, are also vulnerable to slippage by RNA polymerases and the ribosome. Using laboratory evolution techniques, we systematically mapped the occurrence of InDels within a given gene, before and after selection. Our data indicate that frame-shifting InDels were frequently bypassed to give functional proteins at surprisingly high frequencies. Further, we found a strict correlation between the repeat length, the frequency of occurrence of InDels at the DNA level, and the likelihood of bypass by transcriptional/translational slippage. Our results suggest that frame-shifting InDels might comprise functional evolutionary intermediates, and an effective mean of sequence divergence ( e.g. when an adjacent InDel restores the frame, resulting in altered sequence and, potentially, in an altered protein structure).